This invention relates generally to the field of confocal microscopes. In particular, it is related to a.novel class of integrated, miniaturized, fiber-coupled, high-precision confocal scanning endoscopes that are particularly suitable for biomedical applications.
The advent of fiber optics and laser technology has brought a renewed life to many areas of conventional optics. Confocal microscopes, for example, have enjoyed higher resolution, more integrated structure, and enhanced imaging capability. Consequently, confocal microscopes have become increasingly powerful tools in a variety of applications, including biological and medical imaging, optical data storage and industrial applications.
In recent years, a great deal of ingenuity has accordingly been devoted to improving the axial resolution of confocal microscopes. A particularly effective approach is to spatially arrange two separate illumination and observation objective lenses, or illumination and observation beam paths, in such a way that the illumination beam and the observation beam intersect at an angle theta (xcex8) at the focal points, so that the overall point-spread function for the microscope, i.e., the overlapping volume of the illumination and observation point-spread functions results in a substantial reduction in the axial direction. A confocal microscope with such an angled, dual-axis design is termed a confocal theta microscope, or an angled-dual-axis confocal microscope, hereinafter. Its underlying theory is described below for the purpose of elucidating the principle of the present invention. A more detailed theory of the confocal theta microscopy can be found in U.S. Pat. No. 5,973,828; by Webb et al. in xe2x80x9cConfocal microscope with large field and working distancexe2x80x9d, Applied Optics, Vol.38, No.22, pp.4870; and by Stelzer et al. in xe2x80x9cA new tool for the observation of embryos and other large specimens: confocal theta fluorescence microscopyxe2x80x9d, Journal of Microscopy, Vol.179, Part 1, pp. 1; all incorporated by reference.
The region of the point-spread function of a confocal microscope""s objective that is of most interest is the region in which the point-spread function reaches its maximum value. This region is referred to as the xe2x80x9cmain lobexe2x80x9d of the point-spread function in the art. It is typically characterized in three dimensions by an ellipsoid, which extends considerably further in the axial direction than in the transverse direction. This characteristic shape is the reason that the axial resolution is inherently poorer than the transverse resolution in a conventional confocal microscope. When the main lobes of the illumination and observation point-spread functions are arranged to intersect at an angle in a confocal theta microscope, however, a predominantly transverse and therefore narrow section from one main lobe is made to multiply (i.e., zero out) a predominantly axial and therefore long section from the other main lobe. This synergistic multiplication of the two point-spread functions reduces the length of the axial section of the overall point-spread function, thereby enhancing the overall axial resolution.
This enhancement of axial resolution is even more dramatic when this technique is used with relatively low numerical aperture (NA) lenses. The shape of the overall point-spread function can be further adjusted by varying the angle at which the main lobes of the illumination and observation point-spread functions intersect.
In addition to achieving higher resolution, an angled-dual-axis confocal microscope described above renders a number of additional important advantages. For instance, since the observation beam is positioned at an angle relative to the illumination beam, scattered light along the illumination beam does not easily get passed into the observation beam, except in the region where the beams overlap. This substantially reduces scattered photon.noise in the observation beam, thus enhancing the sensitivity and dynamic range of detection. Moreover, the illumination and observation beams do not become overlapping until very close to the focusxe2x80x94this effect is particularly prominent when using low NA focusing elements (or lenses) for focusing the illumination and observation beams. Therefore, such an arrangement prevents scattered light in the illumination beam from directly xe2x80x9cjumpingxe2x80x9d to the corresponding observation beam, thereby further filtering out scattered photon noise in the observation beam. As such, an angled-dual-axis confocal microscope has much lower noise and is capable of providing much higher contrast when imaging in a scattering medium, rendering it highly suitable for imaging within biological specimens.
Furthermore, in recent years optical fibers have been used in confocal systems to transmit light more flexibly. A single-mode fiber is typically used so that an end of the fiber is also conveniently utilized as a pinhole for both light emission and detection. This arrangement is not susceptible to the alignment problems the mechanical pinholes in the prior art systems tend to suffer. Moreover, the use of optical fibers enables the microscopes to be more flexible and compact in structure, along with greater maneuverability in scanning.
The aforementioned angled-dual-axis confocal arrangement can be further utilized to perform two-photon (and multi-photon) fluorescence microscopy, so as to exploit its high resolution and low noise capabilities. Two-photon (and multi-photon) fluorescence microscopy has been described and performed in the art, as exemplified by Lakowicz et al. in xe2x80x9cTwo-color Two-Photon Excitation of Fluorescencexe2x80x9d, Photochemistry and Photobiology, 64(4), (1996) pp.632-635; by Beaurepaire et al. in xe2x80x9cCombined scanning optical coherence and two-photon-excited fluorescence microscopyxe2x80x9d, Optics Letters, Vol.24, No.14, (1999) pp. 969-971; and by Lindek et al. in xe2x80x9cResolution improvement in nonconfocal theta microscopyxe2x80x9d, Optics Letters, Vol.24, No.21, (1999) pp.1505-1507. In such an arrangement, two illumination beams are directed to intersect optimally, such that each of the two observation beams thus produced is in an optimal confocal arrangement with its corresponding illumination beam. Whereas traditional single-photon fluorescence laser microscopy requires only a single photon xcex3 for excitation, two-photon fluorescence microscopy requires simultaneous absorption of two photons xcex1 and xcex2 for excitation. In terms of energy, hc/xcex3=hc/xcex1+hc/xcex2. Thus, xcex1 and xcex2 are both longer in wavelength than xcex3. However, it is important to note that xcex2 need not necessarily equal xcex1. Indeed, any combination of wavelengths can be used, so long as the net energy requirements for exciting a particular type of fluorophores are satisfied. Accordingly, two-photon (or multi-photon) fluorescence microscopy has been used in the art for imaging various types of fluorophores (or fluorophore indicators attached to proteins and biological cells) that are of particular interest to biomedical applications.
The past few years have seen a number of confocal theta microscopes in the art for performing scanning reflectance and fluorescence microscopy, as exemplified by German Patent DE-OS 43 26 473 A1; by Webb et al. in xe2x80x9cConfocal microscope with large field and working distancexe2x80x9d, Applied Optics, Vol.38, No.22, pp.4870; by U.S. Pat. No. 5,973,828 of; by U.S. Pat. No. 6,064,518 of; by U.S. Pat. No. 5,034,613 of Denk et al.; and by U.S. Pat. No. 6,020,591 of Harter et al. None of these prior art confocal systems, however, perform the scanning microscopy in an angled-dual-axis confocal arrangement that is easily scalable to a small size instrument. Moreover, the designs of these prior art confocal systems are such that they do not lend these systems to be miniaturized confocal scanning endoscopes, suitable for biomedical imaging and other applications where relatively long working distance, large field of view, high resolution, fast scanning, and highly compact and maneuverable imaging tools are required.
Hence, there is a need in the art for a miniaturized confocal scanning endoscope that provides high resolution, fast scanning, high sensitivity, and versatile imaging capabilities.
The aforementioned need in the art is provided by a novel class of integrated angled-dual-axis confocal scanning endoscopes according to the present invention. An integrated angled-dual-axis confocal scanning endoscope of the present invention advantageously exploits the benefits of using relatively low NA objectives and a xe2x80x9cpost-objectivexe2x80x9d scanning means in an angled-dual-axis confocal arrangement, and a silicon micro-machined and fiber-coupled construction. The integrated angled-dual-axis confocal scanning endoscope thus constructed provides sufficient resolution, working distance, field of view, scanning speed and sensitivity suitable for biological imaging in a highly integrated and compact structure, rendering it further adaptable to an in-vivo imaging endoscope. One or two illumination beams may be employed in the angled-dual-axis confocal scanning endoscope of the present invention, thereby providing an assortment of reflectance and fluorescence images. The angled-dual-axis confocal scanning endoscope of the present invention is further capable of providing a combination of line and surface scans with fast speed and high precision.
The present invention provides a micro-machined angled-dual-axis confocal scanning endoscope, comprising a silicon substrate, a scanning mirror means mechanically coupled to the substrate, a silicon spacer containing a cavity, a base-plate carrying first and second reflective focusing elements along with an optical window, and first and second optical fibers. The cavity is disposed between the scanning mirror means and the optical window. The cavity is typically produced in the silicon spacer by way of anisotropical etching known in the art of silicon fabrication technology for producing cavities in silicon structure. The cavity thus produced comprises first and second side-walls that are positioned with predetermined and precise orientations, and also bear first and second V-grooves respectively. First and second optical fibers pass through the first and second side-walls by way of the first and second V-grooves, such that each of the first ends of the first and second optical fibers is in direct optical communication with the cavity. The inner surfaces of the first and second side-walls provide first and second reflective surfaces, each with a precise and predetermined orientation, as determined by the well-known crystal structure of silicon and the way of etching taking place in silicon. The first and second reflective focusing elements are generally selected from the group consisting of diffractive optical elements, reflective diffraction lenses, holographic optical elements, reflective off-axis binary lenses, and curved mirrors. The scanning mirror means may be mechanically coupled to the substrate by way of a hinge means, which also provides a pivoting axis. The optical window in the base-plate is disposed adjacent to an object, so as to provide the passages of optical beams between the cavity and the object.
It should be noted that in this specification and appending claims, a scanning mirror means should be construed in a broad sense as including one or more scanning mirrors that can rotate about one or two axes, one or more assemblies of scanning mirrors along with appropriate mechanical/electrical coupling mechanisms that provide rotation about one or two axes, or other scanning means known in the art that can provide beam steering/scanning in one or two dimensions. For instance, a scanning mirror means may comprise a single scanning mirror (such as a silicon scanning micro-machined mirror), or an assembly of two (or more) scanning mirrors arranged such that they jointly provide rotation about one or two orthogonal axes. It can also comprise a gimbaled assembly of a scanning mirror and a frame which are configured to provide rotation about two orthogonal axes, or a combination of two (or more) such gimbaled assemblies. A skilled artisan can devise an appropriate scanning mirror means in accordance with the present invention, for a given application.
In operation, an illumination beam emerges from the first end of the first optical fiber and is directed onto the second reflective surface, which in turn deflects the illumination beam to the first reflective focusing element. The focused illumination beam is then passed onto and further directed by the scanning mirror means through the optical window to a first substantially diffraction-limited focal volume along a first optical axis within an object. Accordingly, an observation beam emanated from a second substantially diffraction-limited focal volume along a second optical axis within the object passes through the optical window into the cavity and is in turn collected by the scanning mirror means. The observation beam is then deflected onto the second reflective focusing element. The focused observation beam is further passed onto the first reflective surface, which in turn directs the observation beam to the first end of the second optical fiber. As such, the assembly of the first and second reflective surfaces along with the first and second reflective focusing elements constitutes an angled-dual-axis confocal focusing means in this case, which provides the first and second optical axes. The first and second optical axes are directed to intersect at an angle xcex8, such that the first and second focal volumes intersect synergistically at a confocal overlapping volume. The scanning mirror means further pivot the illumination and observation beams in such a way that the confocal overlapping volume of the beams moves through the object, thereby producing a scan.
In one embodiment of the present invention, the scanning mirror means comprises a single scanning mirror, which is substantially flat and can rotate about a pivoting axis. The scanning mirror pivots the illumination and observation beams jointly in such a way that the first and second focal volumes remain intersecting synergistically and that the confocal overlapping volume moves along an arc-line within the object, thereby producing an arc-line scan. Such an arc-line scan can also be obtained by using a scanning mirror means comprising two (smaller) scanning mirrors that are substantially co-planar and can co-rotate about a common pivoting axis. These mirrors can be operated in substantially synchronous motion, so as to scan illumination and observation beams in a way functionally equivalent to a single (larger) scanning mirror An advantage of using two smaller scanning mirrors is that faster scanning can be provided.
In another embodiment of the present invention, the scanning mirror means comprise two scanning mirrors that are substantially flat. The two scanning mirrors can rotate about their respective individual pivoting axes, which are substantially parallel. By counter-rotating relative to each other about the respective pivoting axes, the two scanning mirrors pivot the illumination and observation beams in such a way that the first and second focal volumes remain intersecting synergistically and that the confocal overlapping volume progressively deepens into the interior of the object along a vertical line, thereby producing a vertical-line scan.
In an alternative embodiment of the present invention, the scanning mirror means comprises a bi-axial scanning element capable of rotating about two orthogonal axes. Such a bi-axial scanning element can be provided by a gimbaled assembly of a scanning mirror and a frame, wherein the scanning mirror can rotate about a first pivoting axis and the frame along with the scanning mirror can rotate about a second pivoting axis. The first and second pivoting axes are configured to be substantially orthogonal. In this case, by rotating about either of the first or second pivoting axis, the scanning mirror pivots the illumination and observation beams jointly in such a way that the first and second focal volumes remain intersecting synergistically and that the confocal overlapping volume moves along an arc-line within the object, thereby producing an arc-line. Moreover, by rotating about both of the pivoting axes in a raster-scanning or other predetermined fashion, the scanning mirror can cause the confocal overlapping volume to move in a predetermined pattern along an arc-cross-sectional surface within the object, thereby producing an arc-cross-sectional-surface scan. For instance, by rotating the bi-axial scanning element about the first and second pivoting axes (where one pivoting axis provides a fast-scanning-axis and the other pivoting axis provides a slow-scanning-axis) in a raster-scanning manner, a successive sequence of arc-line scans along an arc-cross-sectional-surface can be produced.
In yet another embodiment of the present invention, the scanning mirror means comprises two bi-axial scanning elements, wherein each can be a gimbaled assembly as described above. The two bi-axial scanning elements are configured such that they can co-rotate about a common pivoting axis and thereby produce an arc-line scan, as in the case of a single scanning mirror described above. The two bi-axial scanning elements can further rotate about two individual pivoting axes that are substantially parallel and spaced apart. By counter-rotating relative to each other about the individual pivoting axes respectively, the bi-axial scanning elements can jointly cause the confocal overlapping volume to move in a predetermined pattern along a vertical-cross-sectional plane, thereby producing a vertical-cross-sectional scan. As a way of example, by first co-rotating about the common pivoting axis (fast-scanning-axis) and then counter-rotating about the respective individual axes (slow-scanning-axes) in a raster-scanning fashion, the two bi-axial scanning elements jointly cause the confocal overlapping volume to move in a successive sequence of arc-line scans that progressively deepen into the interior of the object along a vertical-cross-sectional plane. Alternatively, by first counter-rotating about the respective individual axes (fast-scanning-axes) and then co-rotating about the common pivoting axis (slow-scanning-axis) in a raster-scanning fashion, the two bi-axial scanning elements jointly cause the confocal overlapping volume to move in a successive sequence of radial-line scans, wherein each radial-line scan is angularly displaced relative to its adjacent radial-line scans in a fan-like pattern along a vertical-cross-sectional plane within the object.
It should be understood that when describing the intersection of the illumination and observation beams in this specification, the term xe2x80x9csynergisticxe2x80x9d means that the intersection of the first and second focal volumes (i.e., the main lobe of the illumination beam""s point-spread function and the main lobe of the observation beam""s point-spread function) is such that the resulting overlapping volume has comparable transverse and axial extents. This synergistic overlapping volume is termed xe2x80x9cconfocal overlapping volumexe2x80x9d in this specification and appending claims. As such, the illumination beam intersects synergistically with the observation beam in the angled-dual-axis arrangement described above. Moreover, the observation beam described above should be construed in a broad sense as each carrying any light transmitted back from the object, including reflected light, scattered light, and fluorescent light of single-photon, two-photon, and multi-photon type.
A unique feature of the angled-dual-axis confocal scanning endoscope of the present invention is that the scanning mirror means is in direct optical communication with the angled-dual-axis focusing means and the object to be examined. This xe2x80x9cpost-objectivexe2x80x9d type of scanning technique enables the best on-axis illumination and observation point-spread functions to be utilized throughout the entire angular range of an arc-line scan, thereby providing greater resolution over a larger transverse field of view, while maintaining substantially diffraction-limited (or relatively aberration-free) performance. Such an arrangement is made possible by taking advantage of the longer working distance rendered by using relatively lower NA focusing elements in the angled-dual-axis focusing means.
Another important advantage of the angled-dual-axis arrangement of the present invention is that since the observation beam is positioned at an angle relative to its corresponding illumination beam, scattered light along the illumination beam does not easily get passed into the observation beam, except in the region where the beams overlap. This substantially reduces scattered photon noise in the observation beam, thus enhancing the sensitivity and dynamic range of detection. Moreover, by using low NA focusing elements in an angled-dual-axis confocal scanning endoscope of the present invention, the illumination and observation beams do not become overlapping until very close to the focus. Such an arrangement prevents scattered light in the illumination beam from directly xe2x80x9cjumpingxe2x80x9d to the corresponding observation beam, thereby further filtering out scattered (or fluorescent) photon noise in the observation beam. Altogether, the angled-dual-axis confocal scanning endoscope of the present invention has much lower noise and is capable of providing much higher contrast when imaging in a scattering medium than the prior art confocal systems employing high NA lenses, rendering it highly suitable for imaging within biological specimens.
A further distinct advantage of the present invention is evident in the scalability of the angled-dual-axis confocal scanning endoscope, which allows for miniaturization while providing sufficient resolution, field of view, and working distance suitable for in-vivo imaging of biological specimens.
Two illumination beams can be employed in an angled-dual axis confocal scanning endoscope of the present invention, such as in any one of the embodiments described above. In this scenario, first and second illumination beams with first and second wavelengths emerge from the first ends of the first and second optical fibers respectively. The angled-dual-axis focusing means focuses and the scanning mirror means direct the two illumination beams through the optical window along the first and second optical axes, such that they intersect synergistically at a confocal overlapping volume within an object. First and second observation beams thus produced pass through the optical window into the cavity and are in turn collected along the second and first optical axes respectively by the scanning mirror means. The observation beams are then focused into the first ends of second and first optical fibers respectively by way of the angled-dual-axis focusing means. The scanning mirror means further pivot the first and second illumination beams (and thereby their corresponding observation beams) in such a way that the confocal overlapping volume of the beams moves through the object, thereby producing a scan. For instance, by utilizing various types of the scanning mirror means described above, arc-line scans, vertical-line scans, arc-cross-sectional-surface scans, and vertical-cross-sectional scans can be accordingly produced.
The aforementioned first and second illumination beams may have the same wavelength, for instance, in the infrared range. The fluorescence light thus produced would include one-color two-photon and multi-photon types of fluorescence. The first and second illumination beams may also have very different wavelengths. As a way of example, the first wavelength may be in the infrared range, while the second wavelength lies in the visible range. The fluorescence light thus obtained would include two-color two-photon (and possibly multi-photon) fluorescence. A skilled artisan will know how to selectively make use of a particular type of reflected and fluorescence light collected from the object and filter out spurious background light for a given application. (The filtering can be accomplished by way of band-pass filters, dichroic filters, wavelength-selective optical elements and the like, for instance).
In addition to collecting the first and second observation beams, a third observation beam comprising predominantly fluorescence light can be collected, providing an additional avenue for collection and detection of fluorescence light. The third observation beam can be collected by a third focusing element mounted on (or made to be an integral part of) the base-plate, such that it is in direct optical communication with the optical window. The third focusing element further focuses the third observation beam to an input end of a third optical fiber, which may be mechanically coupled to the substrate. It should be noted that the scanning mirror means is not involved in collecting the third observation beam in this case. The third optical fiber is preferably a multi-mode fiber of larger diameter (or made of a bundle of multiple optical fibers), so as to maximize the collection efficiency of light emanating from the confocal overlapping volume throughout its motion within the object during scanning. The first and second optical fibers are preferably single-mode fibers. Alternatively, an optical detector (such as a silicon photon detector) can be mounted on (or made to be an integral part of) the silicon substrate, so as to collect the third observation beam.
As such, an angled-dual-axis confocal scanning endoscope employing two illumination beams is capable of providing an assortment of reflectance and fluorescence images. For instance, a first wavelength-selective-beam-splitting means can be coupled to the first observation beam, diverting a portion of the first observation beam to a first optical detector. The first wavelength-selective-beam-splitting means can be configured to preferentially permit only the reflected light (characterized by a particular wavelength and bandwidth of light) carried by the first observation beam to pass through, thereby providing a first reflectance image signal.
A second wavelength-selective-beam-splitting means can be further coupled to the first observation beam, diverting an additional portion of the first observation beam to a second optical detector. The second wavelength-selective-beam-splitting means may be designed to preferentially permit only the particular wavelength and bandwidth of light corresponding to two-photon fluorescence light carried by the first observation beam to pass through, thereby providing a two-photon fluorescence image signal. Likewise, a third wavelength-selective-beam-splitting means can be coupled to the second observation beam, diverting a portion of the second observation beam to a third optical detector. The third wavelength-selective-beam-splitting means can be configured to preferentially permit only the reflected light (characterized by a particular wavelength and bandwidth of light) carried by the second observation beam to pass through, thereby providing a second reflectance image signal. And a fourth wavelength-selective-beam-splitting means may be further coupled to the second observation beam, providing an additional avenue for detecting the two-photon fluorescence light carried by the second observation beam, and so on. All in all, a cascade of the wavelength-selective-beam-splitter means can be optically coupled to either of the first and second observation beams, enabling various spectral components of each of the observation beams to be extracted and detected. Moreover, a superposition of reflectance and two-photon fluorescence images thus obtained would be highly desirable, for it provides complementary information about the morphology and functionality of a biological sample. It should be noted that it is possible to operate the present invention in a number of ways that would provide different combinations of reflectance and fluorescence (single-photon, two-photon, or multiple-photon) images, depending upon the instrument design and the types of light sources/wavelengths used. It is preferable to design the instrument in a way that maximizes the resolution of the images thus produced and contemporaneously minimizes the scattered and/or fluorescent photon noise in the image signal. This can be best accomplished by the following seven design rules, which insure that reflected or fluorescence light generated by each illumination beam is optimally collected only by its corresponding (angularly overlapping) observation beam:
1) In the case where the first observation beam is being used to collect reflectance image information characterized by a first wavelength, the second illumination beam should not include light with the first wavelength, and the first illumination beam must provide light with the first wavelength.
2) In the case where the first observation beam is being used to collect single-photon fluorescence image information characterized by a third wavelength when the object is excited by light of a second wavelength, the second illumination beam should not include single-photon excitation light with the second wavelength, and the first illumination beam should provide single-photon excitation light with the second wavelength.
3) In the case where the first observation beam is being used to collect one-color two-photon (1C2P) fluorescence image information characterized by a fifth wavelength when the object is excited by light of a fourth wavelength, the second illumination beam should not include 1C2P excitation light with the fourth wavelength, and the first illumination beam should provide 1C2P excitation light with the fourth wavelength.
4) In the case where either of the first and second observation beams, or both of the observation beams, are being used to collect two-color two-photon (2C2P) fluorescence image information characterized by an eighth wavelength when the object is excited by light that requires both of sixth and seventh wavelengths, the first and second illumination beams should each provide light with only one of the two required wavelengths, such that 2C2P excitation light is provided only in the region where the two illumination beams overlap both spatially and temporally.
5) In the case where the second observation beam is being used to collect reflectance image information characterized by a ninth wavelength, the first illumination beam should not include light with the ninth wavelength, and the second illumination beam must provide light with the ninth wavelength.
6) In the case where the second observation beam is being used to collect single-photon fluorescence image information characterized by an eleventh wavelength when the object is excited by light of a tenth wavelength, the first illumination beam should not include single-photon excitation light with the tenth wavelength, and the second illumination beam should provide single-photon excitation light with the tenth wavelength.
7) In the case where the second observation beam is being used to collect one-color two-photon (1C2P) fluorescence image information characterized by a thirteenth wavelength when the object is excited by light of a twelfth wavelength, the first illumination beam should not include 1C2P excitation light with the twelfth wavelength, and the second illumination beam should provide 1C2P excitation light with the twelfth wavelength.
The present invention further provides an angled-dual-axis confocal scanning system, comprising an angled-dual-axis confocal scanning endoscope of the present invention, first and second light sources, and first and second optical detectors. The first light source is optically coupled to a second end of the first optical fiber of the angled-dual-axis confocal scanning endoscope by way of a first wavelength-selective-beam-splitting element, providing the first illumination beam. The first wavelength-selective-beam-splitting element additionally diverts a portion of the second observation beam delivered by the first optical fiber to the first optical detector. Likewise, the second light source is optically coupled to a second end of the second optical fiber of the angled-dual-axis confocal scanning endoscope by way of a second wavelength-selective-beam-splitting element, providing the second illumination beam. The second wavelength selective beam-splitting element additionally diverts a portion of the first observation beam delivered by the second optical fiber to the second optical detector. By selecting appropriate first and second wavelength-selective-beam-splitting elements, various spectral components of the first and second observation beams can be extracted and detected according to the aforementioned design rules
In the aforementioned angled-dual-axis confocal scanning system of the present invention, either of the first and second wavelength-selective-beam-splitting elements can be a dichroic filter, a wavelength division multiplexer (WDM), or a fiber-optic coupler. Each of the first and second light sources can be a continuous wave (CW) or pulsed light source, such as a fiber laser, a semiconductor laser, a diode pumped solid state laser, or other suitable fiber-coupled light source known in the art. The optical detector can be a PIN diode, an avalanche photo diode (APD), or a photo-multiplier tube. Such an angled-dual-axis confocal scanning system provides a simple and versatile imaging tool with high resolution and fast scanning capability.
All in all, the angled-dual-axis confocal scanning endoscopes of the present invention have advantages of higher resolution, faster scanning, higher sensitivity and larger dynamic range of detection, a larger field of view and a longer working distance, a compact and integrated construction, and contemporary reflectance and fluorescence imaging.